Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 200 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protecting against HCV infection and disease have yet to be established.
HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is of positive polarity and comprises one open reading frame (ORF) of approximately 9600 nucleotides in length, which encodes a linear polyprotein of approx. 3010 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. The structural proteins (C, E1, E2 and E2-p7) comprise polypeptides that constitute the virus particle. The non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) encode for enzymes or accessory factors that catalyze and regulate the replication of the HCV RNA genome. Processing of the structural proteins is catalyzed by host cell proteases. The generation of the mature non-structural proteins is catalyzed by two virally encoded proteases. The first is the NS2/3 zinc-dependent metalloprotease which auto-catalyzes the release of the NS3 protein from the polyprotein. The released NS3 protein contains an N-terminal serine protease domain and catalyzes the remaining cleavages from the polyprotein. The released NS4A protein has at least two roles. The first role is forming a stable complex with NS3 protein and assisting in the membrane localization of the NS3/NS4A complex; the second is acting as a cofactor for NS3 protease activity. This membrane-associated complex, in turn catalyzes the cleavage of the remaining sites on the polyprotein, thus effecting the release of NS4B, NS5A and NS5B. The C-terminal segment of the NS3 protein also harbors nucleoside triphosphatase and RNA helicase activity. The function of the protein NS4B is unknown. NS5A is a highly phosphorylated protein that appears to be responsible for the interferon resistance of various HCV genotypes. NS5B is an RNA-dependent RNA polymerase (RdRp) that is involved in the replication of HCV.
The open reading frame of the HCV RNA genome is flanked on its 5′ end by a non-translated region (NTR) of approx. 340 nucleotides that functions as the internal ribosome entry site (IRES), and on its 3′ end by an NTR of approximately 230 nucleotides. Both the 5′ and 3′ NTRs are important for RNA genome replication. The genomic sequence variance is not evenly distributed over the genome and the 5′NTR and parts of the 3′NTR are the most highly conserved portions.
The cloned and characterized partial and complete sequences of the HCV genome have been analyzed with regard to appropriate targets for a prospective antiviral therapy. The following four viral enzyme activities provide possible targets: (1) the NS2/3 protease; (2) the NS3/4A protease complex, (3) the NS3 helicase and (4) the NS5B RNA-dependent RNA polymerase (NS5B RdRp). The NS5B RNA dependent RNA polymerase has also been crystallized to reveal a structure reminiscent of other nucleic acid polymerases (Ago et al. 1999; Bressanelli et al. 1999; Lesburg et al. 1999) with an enclosed active site.
Virus-specific functions essential for replication are the most attractive targets for drug development. The absence of RNA dependent RNA polymerases in mammals, and the fact that this enzyme appears to be essential to viral replication, would suggest that the HCV NS5B polymerase is an ideal target for anti-HCV therapeutics. It has recently been demonstrated that mutations destroying NS5B activity abolish infectivity of HCV RNA in a chimp model (Kolykhalov, A. A. et al. 2000). The initial step of viral RNA replication is recognition of the 3′-end of RNA template by NS5B (RdRp), which may occur directly or indirectly with the help of cellular proteins (Lai, 1998; Strauss et al., 1999). HCV polymerase then proceeds to elongate this template and form a complementary RNA product.
Several groups have described the crystal structure of HCV NS5B polymerase (Ago et al. 1999 supra; Bressanelli et al. 1999 supra; Lesburg et al. 1999 supra). It resembles a flattened sphere with the approximate dimensions 70 Å×60 Å×40 Å. The polypeptide chain encircles the active site, forming a cavity at the center of the molecule, and resulting in an appearance that is very different from other U-shaped polymerases. The domain organization of NS5B is consistent with other polymerases in that it is subdivided into finger, palm and thumb domains in which the palm domain, i.e. residues 188-225 and 287-370, is conserved. In contrast to other polymerases, extensive thumb and finger domain contacts result in a globular-shaped HCV polymerase. These contacts are mediated, in part, by loops that extend from the finger to the thumb domain. Knowledge of the crystal structure of NS5B is useful for structure-based drug design and, indeed, structures of NS5B polymerase/inhibitor complexes have been reported recently (Wang et al. 2003; Love et al, 2003; EP 1 256 628). Non-nucleoside analogue inhibitors were found to bind in a wedge-like fashion to a hydrophobic binding pocket located near the C-terminal region of the polymerase thumb domain. In this study, the enzyme was determined to undergo only minor conformational changes in the enzyme/inhibitor complex. At least two NTP binding sites have been characterized on NS5B, one in the active site palm and a second potential allosteric site on the thumb (O'Farrell et al. 2003; Bressanelli et al. 2002).
Interestingly, Labonté et al. 2002 have reported that a mutation of Leu30 in the N-terminal finger loop of the NS5B affects its polymerase activity and speculate that a local alteration in the structure of the Leu30 mutant is responsible for this decrease in activity. However, the authors are silent on the presence of a binding pocket that is “masked” by the finger loop in its native state and becomes exposed by a mutation or displacement of the Leu30 residue. The discovery of this peculiar binding pocket is the subject-matter of the present invention.
Accordingly, the effort to develop effective treatments to HCV infection can be facilitated by increased knowledge of the structure of enzymes critical to HCV replication, most notably, the NS5B polymerase. An increased knowledge of enzyme structure, particularly when complexed with specific inhibitors, will lead to a means of identifying binding sites in the enzyme, as well as the conformation of enzyme/inhibitor complexes and susceptible residues in the enzyme, knowledge of each of which is critical to the process of drug design and optimization.